H-042. Application of in vivo Expression Technology as a Means to Identify Plant-Induced Rhizobacterial Genes under Suboptimal Temperature Conditions

K. A. Ford, C. L. Patten;
Univ. of New Brunswick, Fredericton, NB, CANADA.

Although the beneficial effect of plant growth promoting rhizobacteria (PGPR) on plants is well established, the molecular basis is not thoroughly understood. We hypothesize that bacterial genes active in planta aid bacteria in colonization and growth promotion of plants, and therefore, identification of plant-induced genes can elucidate novel pathways for growth promotion. Furthermore, we hypothesize that bacterial genes expressed under suboptimal conditions contribute to the growth of the bacterium and plant under stresses such as cold temperatures. Pseudomonas syringae GR12-2, a cold tolerant PGPR, can promote plant growth at 10°C. In this study, IVET, a promoter trapping technique, is being used to identify genes in P. syringae GR12-2 that are induced by cold-stressed plants. The IVET screen used has two components: (1) P. syringae GR12-2 ΔdapB, an essential gene mutant and (2) an IVET expression vector (pIVET) carrying a promoterless dapB-lacZ transcriptional fusion. dapB is involved in biosynthesis of lysine and diaminopimelate, and as expected, P. syringae GR12-2 ΔdapB did not grow in vitro unless supplemented with 50 and 400 μg/ml of lysine and diaminopimelate respectively. Genomic fragments of P. syringae GR12-2 were inserted upstream of dapB in pIVET and integrated into the genome of P. syringae GR12-2 ΔdapB. Complementation of the dapB mutation using pIVET restored growth of P. syringae GR12-2 ΔdapB in planta to approximately 75% of that of the wildtype strain. Promoters active in planta are being identified based on their ability to survive on plant roots at 10°C; cold-plant induced genes will be differentiated from plant induced genes by testing survivors for growth in planta at 25°C. Constitutively expressed genes are differentiated by development of blue colonies in vitro. Completion of our analysis of the resulting sequences will identify cold induced, plant induced and cold-plant induced genes. This research provides insight into the signal exchange occurring between PGPR and host plants within the rhizopshere. Furthermore, the cold-plant induced genes identified in this work will provide a basis for understanding how PGPR promote growth at suboptimal temperatures.